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Administrative data

Description of key information

The result of the mouse LLNA resulted in the substance being classified as a skin sensitier.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 October 2019 (Experimental start) to 29 October 2019 (Experimental completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
A different positive control batch was used. Incubation of precipitates was 10 days, not overnight, sample measurement was on Day 16. Temp (min:18.9°C, max:26.0°C) > 19-25°C. Relative humidity (max:80%) > 30-70%. Deviations had no impact on the study.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Materia, Inc.
- Lot/batch No.of test material: RP229-0714
- Expiration date of the lot/batch: 7 May 2021
- Purity test date: 28 March 2018 (CoA)

RADIOLABELLING INFORMATION (if applicable)
[Methyl-3H]-Thymidine (American Radiolabeled Chemicals Inc.)
- Batch No.190524
- Radiochemical purity: Not stated
- Specific activity: Not specified
- Injection of Tritiated Thymidine (3HTdR): 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR
- Locations of the label: Not specified
- Expiration date of radiochemical substance: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70% Relative Humidity). Swept with N2 and resealed after use to maintain freshness.
- Stability under test conditions: Assumed stable for the duraytion of the test
- Solubility and stability of the test substance in the solvent/vehicle: The selected vehicle was 2-Butanone (MEK). Assumed stable for the duration of the test
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Diluted in 2-Butanone (MEK).
- Final dilution of a dissolved solid, stock liquid or gel: Distilled Tricyclopentadiene (TCPD) at 25%, 10% and 5% (w/v, formulated in MEK) concentrations.
-
FORM AS APPLIED IN THE TEST (if different from that of starting material) : Applied as a solution

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: None specified
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaCrl mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Not specified. Only healthy animals were used, the health status was certified by the veterinarian.
- Age at study initiation: 10 weeks old (main study)
- Weight at study initiation: 19.8g – 23.1g (main study)
- Housing: Type II. polypropylene/ polycarbonate cage.
- Diet (e.g. ad libitum): ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice” produced by ssniff Spezialdiäten GmbH (D-59494 Soest, Germany), and Geldiet Transport (Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France).
- Water: Animals received tap water from the municipal supply from 500 mL bottles, ad libitum.
- Acclimation period: At least 20 days
- Indication of any skin lesions: None, all mice were certified as healthy by a veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 to 25.0°C
- Humidity (%): 30 to 80%
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light):
- IN-LIFE DATES: From: To: 2 to 7 October 2019 (Prelim experimental start and completion). 23 to 29 October 2019 (Main test experimental start and completion).
Vehicle:
methyl ethyl ketone
Concentration:
Distilled Tricyclopentadiene (TCPD) at 25%, 10% and 5% (w/v), formulated in MEK.
No. of animals per dose:
In the main assay, 20 female CBA/CaCrl mice were allocated to five groups, each group comprised four animals:
- three groups of animals received Distilled Tricyclopentadiene (TCPD) at 25%, 10% and 5% (w/v), formulated in MEK.
- a negative control group received the vehicle (MEK) only.
- a positive control group received 25% (w/v) HCA (formulated in MEK).
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Test item solubility using OECD standard vehicles was examined in a Preliminary Compatibility Test. Of those tested 2-Butanone (MEK) was selected as the vehicle for this study. The test item was weighed and formulations prepared daily on a weight:volume basis as % (w/v).

During the Preliminary Irritation / Toxicity Test
The test item did not dissolve in any vehicle at concentrations of 100 or 50% (w/v).
At 25% and 10% (w/v) no mortality or clinical signs were observed. Test item residue or a minimal amount of test item residue was observed on the ears of the 25% and 10% (w/v) groups from Day 1 or Day 2 to Day 3.
No marked body weight loss (>5% reduction) was observed in any dose group.
No increased ear thickness value (>25%) was detected.
The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).
Based on the above observations, the 25% (w/v) dose group was considered acceptable and was selected as highest dose for the main test.

MAIN STUDY
In the main assay, twenty female CBA/CaCrl mice were allocated to five groups, each group comprised four animals:
- three groups of animals received Distilled Tricyclopentadiene (TCPD) at 25%, 10% and 5% (w/v, formulated in MEK) concentrations,
- a negative control group received the vehicle (MEK) only,
- a positive control group received 25% (w/v) HCA (formulated in MEK).

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429

A unique number written on the tail in permanent marker identified each animal. The animal number was assigned on the basis of Citoxlab Hungary Ltd.’s Master File. Cages were marked with identity cards with information including study code, cage number, dose group, sex and individual animal number. The animals were randomised and allocated to experimental groups. The randomisation was checked by computer software using the body weight to verify homogeneity and variability between groups.

TREATMENT PREPARATION AND ADMINISTRATION:
Formulations were applied to the dorsal surface of the ears of the experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3) and animals were maintained for a further 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minutes after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.
There was no mortality or clinical signs observed during the main assay.
No test item related effect was noted on body weight.

EVALUATION OF THE RESULTS
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value ("DPM"). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as "DPN" (DPM divided by the number of lymph nodes) following the industry standard for data presentation.

Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

Interpretation of Results
The test item is regarded as a sensitizer if both the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Acceptability of the test
The Local Lymph Node Assay is considered valid if it meets the following criteria:

- the DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,
- the positive control substance produces a significant lymphoproliferative response increases (SI>3),
- each treated and control group includes at least 4 animals,
- the test item does not cause serious systemic or local toxicity.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical data processing was performed.
Positive control results:
The positive control (25% (w/v) HCA in MEK) produced a significant lymphoproliferative response increases (SI>3)
Larger than normal lymph nodes were observed in the positive control group (relative to those of the control group)
The DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range.
The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA), formulated in the same vehicle as the test item (SI=14.4) demonstrated the appropriate performance of the assay.
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Negative control (MEK)
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
4.3
Test group / Remarks:
5% (w/v) TCPD formulated in MEK
Remarks on result:
other: Positive
Key result
Parameter:
SI
Value:
4.8
Test group / Remarks:
10% (w/v) TCPD formulated in MEK
Remarks on result:
other: Positive
Key result
Parameter:
SI
Value:
5.3
Test group / Remarks:
25% (w/v) TCPD formulated in MEK
Remarks on result:
other: Positive
Key result
Parameter:
SI
Value:
14.4
Test group / Remarks:
Positive control (25% HCA) formulated in MEK
Remarks on result:
other: Positive
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

Test Group Name Measured DPM/group DPM No. Of Nodes DPN Stimulation Index Values
Background (5% (w/v) TCA ) 34 - - - -
Negative control (MEK) 3182 3148.0 8 393.5 1.0
25% (w/v) 16738 16704.0 8 2088.0 5.3
10% (w/v) 15076 15042.0 8 1880.3 4.8
5% (w/v) 13418 13384.0 8 1673.0 4.3
Positive control (25% HCA) 45272 45238.0 8 5654.8 14.4

Trichloroacetic acid (TCA)
2-Butanone (MEK)
HCA 25% = alpha-Hexylcinnamaldehyde
SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.
DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.
In case of individual approach, SI values were calculated from the mean DPN values of the group.


DETAILS ON STIMULATION INDEX CALCULATION
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value ("DPM"). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as "DPN" (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

EC3 CALCULATION
The test item was a liquid which was applied formulated in MEK. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions were considered to be good evidence that Distilled Tricyclopentadiene (TCPD) is a sensitizer. The dose response does not allow a reliable extrapolation to the EC3 value, although it appears to be below 2% (suggesting a Cat.1A).

CLINICAL OBSERVATIONS:
There was no mortality or clinical signs observed during the main assay.

BODY WEIGHTS
No test item related effect was noted on body weight in the main assay.
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Under the conditions of the present assay, Distilled Tricyclopentadiene (TCPD), tested in a suitable vehicle (MEK), was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

The study result triggers the following classification/labelling:
- Regulation (EC) No 1272/2008 (CLP): Cat.1
- GHS (rev. 7) 2017: Cat.1
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The result of the mouse LLNA resulted in the substance being classified as a skin sensitier.